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Notes: The authors of this study investigated the function of the hypervariable region (HVR) present in open reading frame 1 (ORF1) in the hepatitis E virus (HEV) by measuring the diversity of the HVR in HEV samples from acutely infected patients and in epidemiologically related samples. They sequenced HEV HVR PCR products from limited dilution cDNA from 8 patients PCR positive for ORF2 of HEV. HEV RNA was extracted from serum using a commercial kit, and then HEV RNA was amplified using the Access RT-PCR System. A second round of PCR was performed using GoTaq® polymerase. cDNA was generated using random hexamer or appropriate primers in the presence of Recombinant RNasin® Ribonuclease Inhibitor. (4242)

Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT® Coupled Transcription Translation System and [35S] methionine. (3889)

Notes: The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel. (3836)

Notes: The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System. (3977)

Notes: The authors infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples. (3888)

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene® Labeling System. (3791)

Notes: The authors used RT-PCR to quantitate the level of rnpB expression after complementing the Bacillus subtilis rnpB mutant strainSSB318 with wildtype or mutant rnpB genes from S. aureus or B. subtilis. Preliminary experiments with decreasing amounts of RNA template were performed to show that rnpB RT-PCR product yields were linearly dependent on RNA template amounts after 12 PCR cycles. RT-PCR was performed using the Access RT-PCR System. (3794)

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

Notes: The authors linked outbreaks of acute gastroenteritis in Italy and France with consumption of oysters contaminated with Norovirus. In Italy Norovirus RNA was detected in fecal samples from infected individuals using the Access RT-PCR System and primers for the Norovirus polymerase region. (3792)

Notes: The authors produced transgenic wheat plants with low-copy insertion of 1Ax1 and bar genes without the concomitant insertion of vector sequences using particle bombardment. Tissue-specific expression of the 1Ax1 gene, which has an endosperm-specific promoter, was examined using RT-PCR in 21 transgenic wheat lines. Total RNA was isolated from leaf, endosperm, root and inflorescence tissues, then amplified using the Access RT-PCR System. (3793)

Toxicol. Sci.85, 703-712.
Cytochrome P450 1A1 and 1B1 in human blood lymphocytes are not suitable as biomarkers of exposure to dioxin-like compounds: polymorphisms and interindividual variation in expression and inducibility.2005

van Duursen, M.B., Sanderson, J.T. and van den Berg, M.

Notes: The authors examined interindividual differences in cytochrome P450 CYP1A1 and CYP1B1 expression levels in human lymphocytes before and after exposure to dioxins and dioxin-like compounds. CYP1A1 and 1B1 mRNA levels were assessed with semi-quantitative RT-PCR using the Access RT-PCR System. (3432)

Notes: In this study, the SV Total RNA Isolation System was used to isolate total RNA from venom sacs from Naja Atra (asian cobra). The Access RT-PCR System was used to reverse transcribe atratoxin and atratoxin-b cDNA from the isolated RNA. The amplified cDNAs were then cloned into the pGEM®-T vector. (3126)

Notes: The authors examined the ybxI gene in Bacillus subtilis 168 for beta-lactamase activity and penicillin recognition as the primary structure of YbxI was similar to that of beta lactamases. The SV Total RNA Isolation System was used to isolate RNA from an exponential growth phase B. subtilis 168 culture. To determine if yxbI was present in the harvested total RNA, the Access RT-PCR System was used to amplify the yxbI cDNA and the positive control B. subtilisyjbJ mRNA. The products were run on a 1% agarose gel and stained with ethidium bromide. (3073)

Antimicrob. Agents Chemother.47, 27–33.
Contributions of MexAB-OprM and an EmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes2003

Li X-Z., Poole K. and Nikaido H.

Notes: The authors examined the putative Pseudomonas aeruginosa homolog to the E. coli small multidrug resistance gene EmrE and its possible role in P. aeruginosa intrinsic drug resistance. Total RNA was isolated from 1-2ml log-phase or overnight stationary-phase P. aeruginosa PAO1 cultures grown in LB using the SV Total RNA Isolation System. After treating the RNA with DNase, 0.1µg RNA was used in the Access RT-PCR System to measure emrEPae expression and the constitutive rpsL gene. The two amplification products were then analyzed on a 1.7% agarose gel. (3074)

Notes: Total RNA was isolated from immature barley anthers with the RNAgents® Total RNA Isolation System. Total RNA concentration was determined by spectrophotometric analysis. Next, the authors used the PolyATtract® mRNA Isolation System to isolate poly (A)+ RNA from the total RNA samples. The purified poly (A)+ RNA was used as a template in RT-PCR using the Access RT-PCR System to analyze expression of BEN1 and Bnuc1. (2847)

Notes: The authors describe using the Wizard® Genomic DNA Purification kit and the SV Total RNA Isolation System to isolate genomic DNA and total RNA, respectively, from Streptococcus pneumoniae. The researchers also added RNasin® Ribonuclease Inhibitor to purified total RNA before storing it and using it for later RT-PCR reactions performed with the Access RT-PCR System. (2835)

Notes: The Access RT-PCR System was used to generate nuclear gene fragments from Chlamydomonas reinhardtii for use as probes for RNA blot hybridization assays and microarrays. RT-PCR reaction products were cloned into the pGEM®-T Easy Vector for sequence verification and to allow easier manipulation. Details of the generation of microarray slides printed on GAPII glass slides (Corning) are provided. (2694)

Development128, 4705-4714.
The Drosophila daughterless gene autoregulates and is controlled by both positive and negative cisregulation.2001

Smith III, J.E. and Cronmiller, C.

Notes: RQ1 RNase-Free DNase was used to treat RNA samples purified from Drosophila. The DNase-treated RNA samples were then used in real-time RT-PCR to analyze gal4 and Mdh1 reporter construct transcripts driven by in vivo daughterless factor autoregulation during heat shock. (3025)

J. Bacteriol.182, 2753–2760.
2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris.2000

Pelletier, D.A. and Harwood, C.S.

Notes: Total RNA was isolated from Rhodopseudomonas palustrisusing the SV Total RNA Isolation System. The Access RT-PCR System was used to determine the transcriptional organization of the badHIaliBA and badK genes, genes involved in anaerobic benzoate degradation. (2306)

Notes: To demonstrate an interaction of activated CaM KII with the postsynaptic density protein densin-180, a gst-fusion protein of densin was produced and reacted with autophosphorylated CaM KII. The glutathione matrix-bound material was eluted and immunoblotted for phospho-Thr286-CaM KII. The autophosphorylated CaM KII was pulled down on the Glutathione only when the densin-GST fusion was present. The phosphoCaM KII was detected with the Anti-ACTIVE® CaM KII pAb. The clone of densin was generated by RT-PCR of rat forebrain total RNA with the Access RT-PCR System. (0076)

Notes: The authors described a cluster of 8 genes from P. putida that is thought to be involved in benzoate metabolism: benA, benB, benC, benD, benE, benF, benK, and benR. The transcriptional start site of benA was determined using Promega's Primer Extension System-AMV Reverse Transcriptase. Total RNA used in these primer extension reactions was isolated from P. putida cells using the SV Total RNA Isolation System. The transcriptional organization of the benA, benB, and benC genes was determined by RT-PCR using the Access RT-PCR System. (2301)

Notes: Total RNA was isolated from 106 actively growing Jurkat, MAW, OZZ or Thp1 cells with the SV Total RNA Isolation System. The equivalent RNA from 8 x 104 cells was used for RT-PCR with the Access RT-PCR System. (2180)

Notes: Total RNA was isolated from Neiserria meningitidis using the SV Total RNA Isolation System. The relative levels of crgA transcript in crgA mutants and wild type strains was determined by RT-PCR with the Access RT-PCR System. (2309)

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